Korean J Urol.  2014 Aug;55(8):542-550. 10.4111/kju.2014.55.8.542.

Altered Gene Expression Profile After Exposure to Transforming Growth Factor beta1 in the 253J Human Bladder Cancer Cell Line

Affiliations
  • 1Department of Urology, Soonchunhyang University Cheonan Hospital, Cheonan, Korea.
  • 2Department of Urology, Seoul National University College of Medicine, Seoul, Korea.
  • 3Department of Biochemistry, Soonchunhyang University College of Medicine, Cheonan, Korea.
  • 4Department of Urology, Seoul National University Bundang Hospital, Seongnam, Korea. selee@snubh.org

Abstract

PURPOSE
Transforming growth factor beta1 (TGF-beta1) inhibits the growth of bladder cancer cells and this effect is prominent and constant in 253J bladder cancer cells. We performed a microarray analysis to search for genes that were altered after TGF-beta1 treatment to understand the growth inhibitory action of TGF-beta1.
MATERIALS AND METHODS
253J bladder cancer cells were exposed to TGF-beta1 and total RNA was extracted at 6, 24, and 48 hours after exposure. The RNA was hybridized onto a human 22K oligonucleotide microarray and the data were analyzed by using GeneSpring 7.1.
RESULTS
In the microarray analysis, a total of 1,974 genes showing changes of more than 2.0 fold were selected. The selected genes were further subdivided into five highly cohesive clusters with high probability according to the time-dependent expression pattern. A total of 310 genes showing changes of more than 2.0 fold in repeated arrays were identified by use of simple t-tests. Of these genes, those having a known function were listed according to clusters. Microarray analysis showed increased expression of molecules known to be related to Smad-dependent signal transduction, such as SARA and Smad4, and also those known to be related to the mitogen-activated protein kinase (MAPK) pathway, such as MAPKK1 and MAPKK4.
CONCLUSIONS
A list of genes showing significantly altered expression profiles after TGF-beta1 treatment was made according to five highly cohesive clusters. The data suggest that the growth inhibitory effect of TGF-beta1 in bladder cancer may occur through the Smad-dependent pathway, possibly via activation of the extracellular signal-related kinase 1 and Jun amino-terminal kinases Mitogen-activated protein kinase pathway.

Keyword

Cell line; Gene expression; Microarray analysis; Transforming growth factor beta; Urinary bladder neoplasms

MeSH Terms

Antineoplastic Agents/*pharmacology
Cluster Analysis
Gene Expression Profiling/methods
Gene Expression Regulation, Neoplastic/*drug effects
Genes, Neoplasm
Humans
MAP Kinase Signaling System/drug effects/genetics
Neoplasm Proteins/genetics/metabolism
Oligonucleotide Array Sequence Analysis/methods
Reverse Transcriptase Polymerase Chain Reaction/methods
Signal Transduction/drug effects/genetics
Smad Proteins/genetics/metabolism
Transforming Growth Factor beta1/*pharmacology
Tumor Cells, Cultured/drug effects
Urinary Bladder Neoplasms/*genetics/metabolism/pathology
Antineoplastic Agents
Neoplasm Proteins
Smad Proteins
Transforming Growth Factor beta1

Figure

  • FIG. 1 Hierarchical clustering of the gene expression profiles of 7,714 reliable genes (A) and 1,974 genes showing changes ≥2.0 fold in at least 1 array (B). Gene ontology map (C) and classification of 1,974 reliable genes according to GeneSpring 7.1 (D).

  • FIG. 2 Hierarchical clustering of the gene expression profiles of 7,992 reliable genes (A) and 310 statistically significant genes (B).

  • FIG. 3 Cluster analysis of the 310 statistically significant genes according to the time response. These genes were classified into 5 clusters by GeneMaths. Cluster 1 (red); showing increased expression over the whole time periods but more up-regulated after 24 hours sequentially, Cluster 2 (green); showing decreased expression over the whole time periods, Cluster 3 (purple); without showing meaningful changes at 6 or 24 hours but becoming up-regulated at 48 hours, Cluster 4 (blue); without showing meaningful changes at 6 or 24 hours but becoming down-regulated at 48 hours, Cluster 5 (yellow); showing markedly increased expression over the whole time periods but becoming noticeably up-regulated only at 48 hours after transforming growth factor β1 treatment.

  • FIG. 4 Comparison expression profile on micrarray with the fold change on reverse transcription-polymerase chain reaction of the interesting genes.

  • FIG. 5 The expression patterns of the Smad-dependent pathway related genes. Most signal transducers showed elevated expression.

  • FIG. 6 The expression patterns of the MAP kinsae pathway related genes.


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